Not applicable.
This invention relates to the field of medical treatment and diagnosis. More particularly, it relates to the diagnosis and treatment of endometriosis and infertility.
Endometriosis is a gynecologic disorder estimated to occur in 10% of reproductive age women. The disease occurs in up to 50% of infertile females (L. C. Gudice et al., J. Reprod. Med., 43:252 (1998)). Endometriosis is defined clinically as the presence of lesions within the peritoneal cavity. Usually, endometriosis is confined to the pelvic and lower abdominal cavity; however, it has been reported in other areas, including the pleural cavity. These lesions originate from endometrial tissue normally found within the interior lining of the uterus. It is believed that cells from the uterine lining (endometrium) are released via the fallopian tubes into the abdominal cavity through retrograde menstruation. While the precise mechanisms by which the displaced endometrial tissue forms ectopic lesions remain to be elucidated, the initial establishment of this disease is clearly an invasive event.
Current diagnosis of endometriosis usually involves laparoscopy, a surgical procedure, or magnetic resonance imaging, a costly procedure. No markers have been approved for a non-invasive diagnosis. Surgical therapies involve removal or ablation of ectopic lesions and often hysterectomy. Non-surgical therapies involve systemic administration of hormones and hormone antagonists (e.g. gonadotropin-releasing hormone (GnRH) agonists) or progestins (e.g., danazol), which can be accompanied by serious side effects, and can only be administered for a limited time period (P. Y. Lu and S. J. Ory, Mayo Clin. Proc., 70:453 (1995)). Contemporary surgical and non-surgical therapies suffer from significant morbidity and lack of long term efficacy. Therefore, there is an enormous unmet need for the development of non-invasive diagnostics and new pharmacological approaches for the treatment of endometriosis.
Antileukoprotease (ALP) is a low molecular weight protein (xcx9c14 kd). It is also known as secretory leukoprotease inhibitor (SLPI) (Y. Zhang et al., J. Clinical Invest., 99:894 (1997)) and as xcex11-proteinase inhibitor (xcex11-PI) (J. A. Kramps et al., Ann. New York Acad. Sci., 624:97 (1991)). The major substrates of antileukoprotease, in vivo, are elastase and cathepsin G, enzymes which degrade collagen and other components of the extracellular matrix. It is conventionally thought that ALP plays an important role in the defense of epithelial surfaces against proteolytic damage (R. C. Thompson et al., Proc. Natl. Acad. Sci. USA, 83:6692; J. A. Kramps et al., J. Histochem. Cytochem. 29:712 (1986); K. Ohlsson et al., J. Androl., 16:64 (1995)).
ALP has been localized in the human cervix (R. Heinzel et al., Eur. J. Biochem., 160:61(1986)) and the fetal and adult human lung (L. N. Willems et al., Thorax, 43:784 (1988); J. A. Kramps et al., J. Histochem. Cytochem. 29:712 (1981)). ALP has also been purified from the parotid and submandibular glands, saliva, tears and gut (H. Tegner and O. K. Olsson, Hoppe Seylers Z. Physiol. Chem., 358:431 (1977); U. A. Seemuller et al., FEBS Lett., 199(1):43 (1986)) and the porcine endometrium (S. J. Farmer et al., Mol Endocrinol, 4(8):1095-104 (1995)). It is reported to inhibit release of IgE-mediated histamine from nasal mucosa. U. Westin et al. Mediators of Inflammation, 7:217 (1998).
Antileukoprotease is not expressed in non-pregnant human endometrial tissue. B. Casslen et al., Hoppe-Seyler""s Z. Physiol. Chem., 362:953 (1981). However, ALP is expressed in pregnant pig endometrium. K. L. Reed, Biology of Reproduction, 55:469 (1996) and L. Badinga et al, Molec. Repro. and Dev., 38:357 (1994). The expression of ALP in the endometrium is regulated by estrogen and progesterone but these hormones do not effect its expression in the lung. S. J. Farmer et al. (1990) Molec. Endocrino.l 4(8):1095-104. R. C. Simmen et al. (1991) Biol. Reprod. 44(1):191-200. Local application of recombinant ALP has been used to treat cystic fibrosis and pulmonary emphysema. A. S. Rudolphus et al. (1993) Am. Rev. Respir. Dis. 147(2):442-7. N. G. McElvaney (1992) J. Clin. Invest. 90(4): 1296-301.
Nucleic acids encoding antileukoprotease are described in U.S. Pat. No. 5,845,076 (R. Heinzel et al.). Active analogs of antileukoprotease are described in U.S. Pat. No. 5,871,956 (P. K. Bandyopadhyay et al.) and U.S. Pat. No. 5,900,400 (R. C. Thompson et al.). Antileukoprotease has 107 amino acids. Its structure includes two domains. The C-terminal domain has the protease inhibitory activity. S. P. Eisenberg et al., J. Biol. Chem., 265:7976 (1990) and I. Van-Seuningen et al., Biochem. Biophys. Res. Comm., 179:1587(1991).
Extracellular matrix (ECM) degradation by secreted proteases is essential for endometrial functions such as blastocyst implantation and menstruation. M. D. Spuijbroek et al. (1992) Fertil. Steril. 58(5):929-33 showed a basement membrane and ECM degradation product, namely the N-terminal propeptide of type III collagen, to be increased in the peritoneal fluid (PF) of patients with early (subtle) forms of endometriosis which points toward an increased activity of extracellular matrix degrading proteases in this condition. The expression of matrix metalloproteinases (MMPs) MMP-3, MMP-7 and tissue inhibitor of metalloproteinase-1 (TIMP-1) have also been documented in lesions of endometriosis. K. G. Osteen et al. Semin. Reprod. Endocrinol., 14(3):247-55 (1996).
While the precise mechanisms by which displaced endometrial tissue leads to ectopic lesions remain speculative, the initial establishment of this disease is clearly an invasive event requiring the breakdown of the extracellular matrix. Extensive literature supports the role of proteases in the invasive behavior of normal and neoplastic cells.
M. Sillem et al., Fertility and Sterility 66:468 (1996) reported that enzymatic digestion of endometrial fragments and treatment with a proteinase inhibitor, aprotinin, impaired ectopic growth. MMP""s are a family of 12 structurally homologous enzymes having an atom of zinc in their active domain. They are secreted as inactive enzymes and activated upon proteolysis. Y. Zhang et al., J. Clinical Invest., 99:894 (1997) reported that antileukoprotease inhibited production by cultured monocytes of prostaglandin H synthase-2, which was accompanied by a decrease in production of PGE2, resulting in suppression of matrix metalloproteinases MMP-1, a collagenase, and MMP-9, a gelatinase. K. L. Bruner et al., Metalloproteinases and Experimental Endometriosis, 99:2851 (1997) reported that steroidal suppression of matrix metalloproteinases MMP-3, a stromelysin, and MMP-7, a matrilysin, in human endometrial tissue inhibited establishment of ectopic lesions by the tissue in nude mice. Matrix metalloproteinases also have been implicated in promoting the invasive behavior of cytotrophoblast cells into the uterine endometrium. P. Bischof et al., J. Reprod. Immunol. 39:167 (1998)
We have discovered that antileukoprotease (ALP) is differentially expressed (over-expressed by about 20-fold) in eutopic endometrial tissue of endometriosis patients compared with ectopic tissue in such patients. We used a 10,000 gene oligonucleotide array to compare gene expression in eutopic and ectopic endometrium from patients with endometriosis. This study resulted in a catalog of 1,988 genes that are differentially expressed between eutopic and ectopic endometrium. There were 726 genes expressed at higher levels in the ectopic endometriotic lesions when compared to the eutopic endometrium and 1,262 genes expressed at higher levels in the eutopic endometrium when compared to ectopic lesions. Of these genes, antileukoprotease, a serine protease inhibitor, was expressed 25 times less in ectopic lesions than in the eutopic endometrium from endometriosis patients. It is believed that the action of elastase and cathepsin G contributes to endometriosis by promoting the invasion of ectopic endometrial tissue into tissue in the peritoneal cavity. Accordingly, this invention provides methods for diagnosing endometriosis by detecting elevated levels of ALP in eutopic endometrium, and methods of treating endometriosis by providing a mammalian subject with an amount of ALP effective to inhibit ectopic implantation of endometrial fragments. In one embodiment, this amount is an amount effective to inhibit the activity of elastase and cathepsin G in the peritoneum.
In one aspect this invention provides a method for the treatment of endometriosis in a subject comprising administering to the subject an amount of antileukoprotease effective to inhibit the activity of elastase or cathepsin G, thereby inhibiting ectopic implantation of endometrial fragments.
In another aspect this invention provides a method comprising measuring an amount of antileukoprotease activity in an endometrial tissue sample from a subject.
In another aspect this invention provides a method for diagnosing endometriosis comprising: a) measuring a test amount of antileukoprotease activity in a eutopic endometrial tissue sample from a subject, and b)comparing the test amount with a normal amount of antileukoprotease activity; whereby a test amount above the normal amount provides a positive sign in a diagnosis of endometriosis.
In another aspect this invention provides a method for diagnosing endometriosis comprising: a) measuring a test amount of antileukoprotease activity in an ectopic endometrial tissue sample from a subject, and b) comparing the test amount with a normal amount of antileukoprotease activity; whereby a test amount below the normal amount provides a positive sign in a diagnosis of endometriosis.
In another aspect this invention provides a method for diagnosing endometriosis comprising: a) measuring a first test amount of antileukoprotease activity in an ectopic endometrial tissue sample from a subject, b) measuring a second test amount of antileukoprotease activity in a eutopic endometrial tissue sample from a subject, and c) determining a test difference between the first test amount and the second test amount; d) comparing the test difference with a normal difference between amounts of antileukoprotease activity in ectopic and eutopic endometrial tissue; whereby a test difference greater than a normal difference provides a positive sign in a diagnosis of endometriosis.
In another aspect this invention provides a method for identifying a modulator of antileukoprotease activity comprising: a) contacting a test compound which is not an elastase or cathepsin G modulator with antileukoprotease; b) measuring an amount of antileukoprotease activity; and c) determining whether the measured amount is different than a control amount of activity when antileukoprotease is not contacted with the agent; whereby a measured amount different than a control amount identifies the compound as a modulator of antileukoprotease.